Isolation and identification of lactic acid and non-acid lactic bacteria from “dèguè” of Western Africa traditional fermented millet-based food

Déguè is a traditional fermented millet-based food which is consumed in Burkina Faso and other countries of West Africa. A total of 125 strains of bacteria were selected from 16 samples of déguè . Isolates were studied by determination of morphological and biochemical characteristics. Among the 125 strains of lactic acid bacteria selected, 68 strains were identified as Lactobacillus and 57 strains as Lactococcus. The representative species of the Lactobacillus were: Lactobacillus plantarum (25.6%), Lactobacillus delbrueckii subsp delbrueckii (11.2%), Lactobacillus acidophilus (7.2%), Lactobacillus brevis (4.8%), Lactobacillus buchneri (8%). Lactobacillus cellobiosus (4%), Lactobacillus pentosus (2.4%), Lactobacillus crispatus (1.6%), Lactobacillus fermentum (1.6%), Lactobacillus curvatus (0.8%), Lactobacillus paracasei subsp paracasei (0.8%). Among the 57 strains of lactic acid coccus isolated predominated Pediococcus damnosus strains (14.4%), followed by Lactococcus lactis subsp lactis (6.4%), Pediococcus pentosaceus (3.2%), Pediococcus sp. (3.2%), Leuconostoc lactis (1.6%), Leuconostoc mesenteroides subsp mesenteroides (0.8%), Pediococcus acidilactici (0.8%), Lactococcus curvatus (0.8%) and Tetragenococcus halophilus (0.8%). Many Gram negative bacteria were also isolated as coliforms and proteolic strains that can play a negative contribution on the quality of dèguè.


INTRODUCTION
The finger millet (Eleusine corocana) is an important food crop in arid and semi-arid regions of the world. In Burkina Faso Finger millet production represents a third of the total consumption of food cereals. Pearl millet Pennisetum glaucum is native to the tropical region of western Africa where it is meeting wide all the cultivated and wild varieties (Hama et al., 2009).
These crops are used in production of many traditional fermented products in African and Asian countries (Kumar et al., 2010). Africa is a source of production of *Corresponding author. E-mail: somdasmarius@yahoo.fr.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License traditional fermented foods and is perhaps the continent with the richest variety of lactic acid fermented foods (Franz et al., 2014).
During fermentation, microorganisms contribute to the development of characteristic properties such as taste, aroma, visual appearance, texture, shelf life and safety. The microbiota of fermented foods is dominated by lactic acid bacteria (LAB), which contribute to their nutritional and sanitary qualities (Nout and Motarjemi, 1997).
Lactic acid bacteria play an important role in traditional fermented foods consumed in different countries (Oguntoyinbo and Narbad, 2012).
The lactic acid fermentation is a process which not only improves the organoleptic and hygienic quality but also the nutritional quality in food; it allows especially the good preservation (Ampe et al., 1999). Other microorganisms as Enterobacteriaceae, yeasts and mould were isolated in a lot of based-cereals fermented foods and can influence their quality (Ashenahi, 1994).
LAB may have probiotic characteristics (Lei and Jakobsen, 2004). These bacteria first have to be selected for their ability to survive passage through the gastrointestinal tract.
Probiotic functions may also be associated with other functions that are of interest for nutrition. This is of particular interest for at-risk popula-tions such as pregnant women and young children in developing countries. For instance, the amylase activity of some LAB helps increase the energy content of gruels for the complementary feeding of young children through partial hydrolysis of starch in the food matrix (Songre- Ouattara et al., 2008) but also helps sustain the growth of the microbiota of starchy foods (Tou et al., 2006). Other functions for example, folate and riboflavin synthesis may improve the quality of the food matrix and may be beneficial for the host.
Folate deficiency can lead to neural tube defects, early spontaneous abortion, and megaloblastic anemia, while riboflavin deficiencies can result in growth failure, inflammation of the skin, or vision deterioration (Rohner et al., 2007).
LAB capable of producing B vitamins could be used for fortification of cereal-based foods (Iyer and Tomar, 2009) and as probiotics (Rossi et al., 2011). In this way, bacteria that combine different functional characteristics could be useful for developing improved or new foods made from local raw materials that target specific nutritional needs and health issues.
Many studies have focused on the phenotypic diversity of the LAB in the tropical fermented foods but few works were made on the dèguè, which plays nevertheless an important role in the food in the Burkina Faso. The objective of this work was to isolate, characterize and identify some bacteria occurring in the déguè samples. This information could contribute to the better knowledge of the microbiota of this kind of food, and perhaps the development of starter cultures with predictable characteristics for use in small-scale and commercial production of déguè

Sampling
Sixteen samples of « déguè » olded from one to three days were obtained from local households of Ouagadougou (Burkina-Faso). Samples were carried out in an ice box for microbial analysis in the Laboratory of Microbiology and Biotechnology (Department of Biochemistry and Microbiology, University of Ouagadougou).

Isolation of lactic acid bacteria (LAB)
The 10 -1 dilution was made by diluting 10 g of each dèguè sample in 90 ml of sterile peptone saline water (10 g of peptone, 5 g of NaCl and 1000 ml of water). Further 10-fold serial dilution, ranging from 10 -2 to 10 -7 was done.
LAB were isolated in two media: Man, Rogosa and Sharpe (MRS) and Rogosa agar. Spreed-plated MRS and ROGOSA were incubated anaerobically using BBL Gas Pak plus Anaerobic System, Beckon Dickinson Microbiology System (Cockeysville, MD, USA) at 30°C for 48 h. Unit forming colonies were randomly picked from plates at higher dilution 10 -6 and transferred into 10 ml in test tubes with sterile MRS broth. Pure cultures were made. The isolates were Gram-stained and tested for catalase and oxydase reaction (Harrigan and McCance, 1990). Presumptive LAB were selected based on the morphology, Gram reaction and the catalase test.

Isolation of total coliform (non-LAB)
Coliforms were isolated using the same dilutions, plated on Plate Count agar and Violet Red Bile Lactose Agar, and cultivated for 24-48 h at 37˚C.

Characterization and identification of isolated LAB and non-LAB
The carbohydrate fermentation profiles of LAB isolates were investigated using API 50CH strips and API CHL medium according to manufacturer's instructions (API system, Bio-Merieux, France).
Phenotypical identification of non-LAB was done by the API 20 E gallery. Strains were designated to species using APILAB PLUS (Version 3.33, Bio-Merieux) and standard taxonomic descriptions.

Morphological and biochemical characteristics of the LAB strains
All 125 selected isolates were Gram-positive, non -motile, catalase-oxydase negative and non-spore forming bacteria, they occurred in short rods and in cocci, singly, in pairs or short chains. These characteristics according to Holzapfel (1997) and Bergey's Manual of Systematic Bacteriology indicate lactic acid bacteria. The results are shown in Figure 1.
L. plantarum (25.6%) was more frequently isolated than other species of LAB in household dèguè. It has been observed that the presence of L. plantarum in the cheese (Cameros) from goat's milk decreased the number of fecal coliforms and other enterobacteria in the final product. Indeed LAB starters can contribute to reducing spoilage problems encountered in this domestic fermentation.
Using Api 50 CHL galleries allowed us to find certain strains capable to ferment some complex sugars. For  (2) and L. crispatus (2). Amylolytic LAB contributes in pH reduction in the medium inhibiting the growth of some pathogenic microorganisms such some faecal coliforms.
The fecal streptococci isolated from the dèguè and identified by APILAB PLUS represent 10.60% of non-LAB isolates. The presence of fecal streptococci shows fecal contamination origin of samples. That can be explained by the lack adequate hygiene.
Acinetobacter and Pseudomonas are characterized by significant proteolytic and lipolytic activities. The proteolysis leads to the formation of free amino acids then to decarboxylation or desamination reactions. The volatile amines and ammonia formed are responsible of unpleasant odors and savors in food. Lipolysis leads to the release of free fatty acids modifying the gustatory properties and leading to rancid taste.
(1.51%), Sphingobacterium multivorum (1.51%) and Chryseomonas luteola (1.51 %). The presence of E. coli and other coliforms in the samples indicate that manipulations of these foods were not made in good sanitary conditions.

Conclusion
This study brings out that dèguè contain several microorganisms, lactic acid bacteria and non-lactic acid bacteria. L. plantarum was the lactic acid bacteria species isolated in higher numbers. Many proteolitic species was observed and could influence the quality of the product.

Author's contributions
The present study was carried out in collaboration with all authors. All authors participated in drafting and revising the manuscript. They also read and approved the final manuscript.